Many approaches have been used to identify nucleic acids within cells and tissues. In situ PCR (ISPCR) is extremely sensitive as this technique has been shown to detect single copy DNA and low abundance RNA. ISPCR, however, yields no information on the starting target copy number and thermal amplification of cells increases cellular autofluorescence five-fold. Conversely, in situ hybridization allows quantification of the starting copy number though the sensitivity ranges between 100-1000 copies. Further, the current art of in situ hybridization uses compounds such as dextran sulfate, acetic anhydride, polyethylene glycol (PEG), hydrochloric acid, and others that either fluoresce or increase cellular autofluorescence. In sum, compounds that either fluoresce in the range of the reporter fluorescent dye or increase cellular autofluorescence decrease signal to noise (SNR) and sensitivity. The present invention provides a method to detect and quantify intracellular nucleic acids with a sensitivity between 3-100 copies. This level of sensitivity is acheived by using a novel combination of non- or weakly crosslinking fixatives and exclusion of autofluorescent compounds commonly used for in situ hybridization. In addition, this method allows simultaneous analysis of cell surface markers including but not limited to phenotypic markers, activation markers, functional markers, and antigens associated with cell death injury.
The optimal detection system should be able to detect a very few copies of a particular target with a broad, linear range for quantification. In addition, this detection scheme should allow simultaneous multiparameter (immunophenotypic) analysis and should be adaptable for use on multiple detection platforms (flow cytometer, image analysis). Last, this optimal test should be easy to perform with high throughput capabilities. The most important determinants of successful in situ hybridization experiments are access to target and signal to noise ratio (SNR). Access to intracellular targets, whether protein or nucleic acids, has always been a challenge. In addition, proteins bound to nucleic acids provide additional obstacles for in situ detection. The approaches to overcome these obstacles depend on the cells or tissue. Cells in suspension or adhered to slides are generally intact. Access to nucleic acids in cells involves permeabilization of the cell membrane and removal of protein bound to nucleic acids. Many agents have been used to permeabilize and many have been commercialized as "fix and perm" combinations. In the past, methanol was used to extract lipids, protease were used to digest membrane associated proteins, and saponin was used to extract membrane associated cholesterol. Methanol, however, was a poor fixative and protease treatment was temperamental with a fine line between optimal use and complete obliteration of cells, and saponin was required in all solutions following the fixation step to maintain permeability.
The classic model systems illustrating sensitivity (high SNR) of detection schemes are human papilloma virus infection and HIV infection. The human papilloma virus (HPV) infected cell lines, SiHa and Caski, contain different number of HPV copies. SiHa cells contain two copies of HPV DNA and Caski cells contain about 300 copies of HOV DNA. In situ hybridization can detect HPV DNA in Caski cells but not SiHa cells. In situ PCR, on the other hand, can detect HPV DNA in both cell lines. In situ PCR, however, is inconsistent, technically difficult, and has a low throughput.
Similarly, the HIV life cycle in cells presents the ultimate challenge for gene detection. Determinants of viral replication including expression of unspliced HIV mRNA and plasma free virus has led to the use of virologic markers as a measure of disease status and therapeutic efficacy. A marked increase in the ratio of unspliced to spliced HIV mRNA, as might occur during the shift from latent to productive infection, precedes precipitous drops in CD4 count. Plasma viral load has been shown to correlate with disease progression and has been used to determine HIV kinetic in vivo. These measurements, however, fail to provide information on the cell type of origin, a weakness considering, the effect of HIV gene expression on cell function, the role of infected cells in transmission and dissemination, and the therapeutic potential of blocking cell-type specific coreceptors.